By generating plasmin, plasminogen activators play a central role in dissolving fibrin thrombi. A decade ago streptokinase (SK), an indirect plasminogen (Pgn) activator, was shown to save the lives of patients with myocardial infarction, a finding that revolutionized medical care and underlined the importance of the plasmin(ogen) system. More recent studies have sounded a grimmer note by showing that SK and recombinant tissue Pgn activator (rt-PA) cause full early thrombolytic reperfusion in only 33-55 percent of patients treated. Because full reperfusion could almost half the death rate from MI, and markedly reduce patient disability, there is a clear need to better understand the molecular interactions that regulate the plasmin(ogen) system. Because of its key physiologic and therapeutic importance, our long term goal is to help elucidate the protein-protein interactions that regulate and modify the activity of the plasmin(ogen) system. The indirect plasminogen activator SK is one of the most biologically and medically important of these interacting proteins. Through a series of elegant and complex interactions SK converts Pgn (without cleavage) into the most catalytically efficient Pgn activator known. At least three functional steps can be discriminated in this process: 1) SK forms a tight stable activator complex with Pgn or plasmin, 2) SK generates or unmasks the latent active site in Pgn creating a virgin enzyme (Pgn*), and 3) SK changes the substrate specificity of Pgn* or plasmin so that the complex can cleave plasminogen molecules. This proposal is directed towards dissecting this process of indirect Pgn activation by SK, in order to identify structural elements in SK necessary for active site generation and Pgn substrate processing, to understand the modulatory role of the amino terminus of SK in Pgn activation, and to fully define SK's cleavage and inactivation during Pgn activation. The Specific Aims are: I. To elucidate the cleavage and inactivation of SK during plasminogen activation in order to identify the minimal structural domains of SK which are necessary for activity and to understand the potential functional limitations of SK during fibrinolysis. II. To understand the role of the amino terminal peptide of SK (SK1-59) in modulating the efficiency of indirect Pgn activation by SK. To determine whether SK1-59 can be used to regulate or target the fibrinolytic effects of the SK-PAC. III. To identify the specific structural elements necessary for a) unmasking or generating the active site in Pgn, and b) for creating a substrate binding site for Pgn cleavage. It is anticipated that insights into this process of indirect plasminogen activation will significantly enlarge our understanding of how the catalytic function and substrate specificity of the Pgn-plasmin system are regulated and, how that system may be altered toward therapeutic ends. Given that many of the experimental questions addressed in these studies are also relevant to the efficacy of SK as a therapeutic agent (e.g., its cleavage and inactivation, the regulation of its catalytic efficiency), we are hopeful that the insights gained from these studies many find application in the design of better therapeutic molecules, or towards improving the use of the indirect Pgn activators for the treatment of thrombosis.